Accepted Article Preview: Published ahead of advance online publication
Neuropsychopharmacology www.neuropsychopharmacology.org
Methylphenidate modifies the motion of the circadian clock Lamotrigine in mood disorders and cocaine dependence Cortical glutamate in postpartum depression
Pharmacological Selectivity within Class I Histone Deacetylases Predicts Effects on Synaptic Function and Memory Rescue Gavin Rumbaugh, Stephanie E Sillivan, Emin D Ozkan, Camilo S Rojas, Christopher R Hubbs, Massimiliano Aceti, Mark Kilgore, Shashi Kudugunti, Sathyanarayanan V Puthanveettil, J David Sweatt, James Rusche, Courtney A Miller
Cite this article as: Gavin Rumbaugh, Stephanie E Sillivan, Emin D Ozkan, Camilo S Rojas, Christopher R Hubbs, Massimiliano Aceti, Mark Kilgore, Shashi Kudugunti, Sathyanarayanan V Puthanveettil, J David Sweatt, James Rusche, Courtney A Miller, Pharmacological Selectivity within Class I Histone Deacetylases Predicts Effects on Synaptic Function and Memory Rescue, Neuropsychopharmacology accepted article preview 3 April 2015; doi: 10.1038/npp.2015.93. This is a PDF file of an unedited peer-reviewed manuscript that has been accepted for publication. NPG are providing this early version of the manuscript as a service to our customers. The manuscript will undergo copyediting, typesetting and a proof review before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply.
Received 11 December 2014; revised 25 March 2015; accepted 31 March 2015; Accepted article preview online 3 April 2015
©
2015 Macmillan Publishers Limited. All rights reserved.
Pharmacological selectivity within Class I Histone Deacetylases predicts effects on synaptic function and memory rescue Gavin Rumbaugh1*#, Stephanie E. Sillivan1,2#, Emin D. Ozkan1, Camilo S. Rojas1, Christopher R. Hubbs1, Massimiliano Aceti1, Mark Kilgore3, Shashi Kudugunti4, Sathyanarayanan V. Puthanveettil1, J. David Sweatt3, James Rusche4, Courtney A. Miller1,2* 1
2
Department of Neuroscience and Metabolism & Aging, The Scripps Research Institute, Jupiter, FL USA 3 Department of Neurobiology, The Evelyn F. McKnight Brain Institute, The University of Alabama at Birmingham, Birmingham, AL USA 4 The Repligen Corporation, Waltham, MA USA
# Equal contribution *Correspondence: Gavin Rumbaugh Courtney A. Miller ([email protected]) ([email protected])
Running Title: Memory rescue by synaptogenic Class I HDAC inhibitors
©
2015 Macmillan Publishers Limited. All rights reserved.
ABSTRACT Histone deacetylases (HDACs) are promising therapeutic targets for neurological and psychiatric disorders that impact cognitive ability, but the relationship between various HDAC isoforms and cognitive improvement is poorly understood, particularly in mouse models of memory impairment. A goal shared by many is to develop HDAC inhibitors with increased isoform selectivity in order to reduce unwanted side effects, while retaining procognitive effects. However, studies addressing this tack at the molecular, cellular and behavioral level are limited. Therefore, we interrogated the biological effects of Class I HDAC inhibitors with varying selectivity and assessed a subset of these compounds for their ability to regulate transcriptional activity, synaptic function and memory. The HDAC 1, -2, -3 inhibitors, RGFP963 and RGFP968, were most effective at stimulating synaptogenesis, while the selective HDAC3 inhibitor, RGFP966, with known memory enhancing abilities, had minimal impact. Furthermore, RGFP963 increased hippocampal spine density, while HDAC3 inhibition was ineffective. Genome-wide gene expression analysis by RNA sequencing indicated that RGFP963 and RGFP966 induce largely distinct transcriptional profiles in the dorsal hippocampus of mature mice. The results of bioinformatic analyses were consistent with RGFP963 inducing a transcriptional program that enhances synaptic efficacy. Finally, RGFP963, but not RGFP966, rescued memory in a mouse model of Alzheimer’s Disease (AD). Together, these studies suggest that the specific memory promoting properties of Class I HDAC inhibitors may depend on isoform selectivity and that certain pathological brain states may be more receptive to HDAC inhibitors that improve network function by enhancing synapse efficacy.
©
2015 Macmillan Publishers Limited. All rights reserved.
INTRODUCTION Manipulation of gene transcription through epigenetic modifications to DNA and chromatin represents a promising and broad therapeutic avenue for the treatment of central nervous system disorders, including disorders of cognitive ability, such as Alzheimer’s Disease (AD). AD is marked by increasing dementia as neurodegeneration progresses, starting with the loss of synapses (Terry et al., 1991), which are fundamental components of the brain’s memory storage system. Epigenetics has received particular attention in recent years for its role in memory (Day and Sweatt, 2011) and hopes are particularly high for the potential of epigenetic modulation to drive cognitive enhancement and, perhaps, to rescue memory in AD (Abel and Zukin, 2008; Mikaelsson and Miller, 2011; Rudenko and Tsai, 2014).
Chromatin’s core histone proteins undergo posttranslational modification with memory, including acetylation, providing some molecular clues to how these enzymes regulate cognition. Transcriptionally permissive histone acetylation is regulated by histone acetyltransferases (HATs), which add acetyl moieties and histone deacetylases (HDACs), which remove them. There are five classes of transcriptionally repressive HDACs. Class I (HDAC1, 2, 3 and 8), Class IIA (HDAC 4, 5, 7 and 9), Class IIB (HDAC 6, 10), and Class IV (HDAC11) are zinc-dependent, whereas Class III sirtuins (SIRT1-7) are NAD-dependent and can also participate in non-histone protein acetylation. Numerous labs have now demonstrated that pharmacologic inhibition of HDACs, which leads to an accumulation of lysine acetylation and transcriptional activation, is capable of modulating memory (Graff and Tsai, 2013; Levenson et al., 2004). This has driven an excitement in the field for the potential of memory rescue in the face of disorders characterized by widespread synapse loss and memory impairments. However, the eleven non-sirtuin HDACs are involved in a myriad of processes throughout the body, leading many to propose that increasing HDACi isoform
©
2015 Macmillan Publishers Limited. All rights reserved.
selectivity may improve specificity for a given brain disorder and simultaneously decrease unwanted off-target effects (Graff and Tsai, 2013; Haggarty and Tsai, 2011; Mikaelsson and Miller, 2011). This naturally begs the question of which isoform(s) represent the ideal target for treating memory disorders.
Class I HDACs have received the most attention in relation to cognition. For instance, genetic, embryonic deletion of HDAC2, but not HDAC1, was found to enhance memory in wildtype mice and furthermore, viral-mediated knockdown of HDAC2 in the hippocampal CA1 region was sufficient to rescue memory in a mouse model of neurodegeneration (Graff et al., 2012; Guan et al., 2009). Similar memory enhancement was found following postnatal forebrain deletion of HDAC2 using a CAMKII-Cre driver (Morris et al., 2013). Likewise, focal deletion of HDAC3 accomplished through viral delivery of Cre into the hippocampus of healthy adult HDAC3 floxed mice enhanced memory (McQuown et al., 2011), though the role of this isoform has not been tested in mouse models of cognitive dysfunction. While broad genetic deletion of an HDAC may be accompanied by various developmental compensations and is therapeutically impractical, more temporally restricted manipulations of HDAC activity can be achieved through pharmacologic targeting with small molecules. These manipulations may also be more specific compared to genetic deletion because they target enzymatic activity, rather than deleting the protein. There are at least three classes of engineered HDACi’s: carboxylic acids (e.g. sodium butyrate [NaB], sodium phenylbutryate [SPB]), hydroxamic acids (e.g. trichostatin A [TSA], suberoylanilide hydroxamic acid [SAHA] and crebinostat), and ortho-aminoanilines or benzamides (e.g. RGFP136). However, a pharmacologic approach to HDAC inhibition has historically meant sacrificing isoform selectivity. For instance, TSA, which can enhance memory when delivered to a healthy brain (Blank et al., 2014; Fischer et al., 2007; Hawk et al., 2011;
©
2015 Macmillan Publishers Limited. All rights reserved.
Monsey et al., 2011), targets all 11 HDACs. SPB and valproic acid, HDACi’s with similarly broad activity, rescue memory in mouse models of AD (Kilgore et al., 2010; Ricobaraza et al., 2009). Additional insight into the role of individual HDACs in cognition can be gleaned from HDACi’s with greater isoform specificity. For example, the most commonly used HDACi for cognitive studies, NaB, was once thought to be a broad spectrum inhibitor, but has since been determined to have a surprising degree of selectivity for Class I HDACs (Kilgore et al., 2010). Further, SAHA and crebinostat’s activity are largely limited to Class I HDACs (1, 2, 3 and 8) and HDAC6. Acute administration of NaB, SAHA and crebinostat have all been shown to enhance memory in wildtype mice and to drive synaptogenesis (Blank et al., 2014; Fass et al., 2013; Fischer et al., 2007; Guan et al., 2009; Intlekofer et al., 2013; Levenson et al., 2004; Mahan et al., 2012). Further, NaB and SAHA rescue memory in mice with reduced cognitive ability, including AD models (Fischer et al., 2007; Kilgore et al., 2010). Two of the most selective HDACi’s used to date, RGFP136 and RGFP966, bear a high degree of selectivity for HDAC3 and enhance memory in wildtype mice (Malvaez et al., 2013; McQuown et al., 2011). When the results of these studies are taken together, it can readily be concluded that manipulating members of Class I HDACs (1, 2, 3 and 8) can improve memory, with genetic manipulations further pointing to the potential of HDAC2 and HDAC3 as targets for improving cognition in a “diseased” state. However, selective inhibitors of HDAC2 or HDAC3 have not been tested in mouse models of reduced cognitive function. Furthermore, the impact of selective inhibitors on synaptic function and spinogenesis, which are the neurobiological substrates believed to drive improved cognition in AD models (Knobloch and Mansuy, 2008; Nistico et al., 2012; Pozueta et al., 2013), remains unknown. Therefore, we assessed the impact of several new HDACi’s with differing Class I selectivity profiles at various neurobiological levels to better understand the relationship between HDACi isoform selectivity and regulation of cognitive function.
©
2015 Macmillan Publishers Limited. All rights reserved.
MATERIALS AND METHODS For additional details, please see Supplementary Materials and Methods.
Animals: 8-week old C57BL/6 mice were used for gene expression analyses (The Jackson Laboratory, Bar Harbor, ME). Thy1-GFP(m) (B6.Cg-Tg(Thy1-EGFP)MJrs/J; stock number 007788) and APPswe/PS1dE9 double transgenic mice (APP/PS1; B6C3-Tg (APPswe,PSEN1dE9)85Dbo/J; stock number 004462) (Jankowsky et al., 2003) were also obtained from Jackson Laboratory. P0 CD1 mice were used for electrophysiological recordings and synaptogenesis assays (CD1(ICR) (Strain Code 022), Charles River Laboratory, Hollister, CA). All procedures were performed in accordance with the Scripps Florida Research Institute at Jupiter, Florida Institutional Animal Care and Use Committee and with national regulations and policies.
Drugs: Trichostatin A (TSA; Tocris) was dissolved in DMSO at a concentration of 300mM. RGFP966, RGFP963, and RGFP233 were provided by Repligen Corporation. The drugs were dissolved in DMSO and diluted in 30% HPBCD, 0.1M acetate, pH 5.5. The final DMSO concentration was no greater than 5% and the same concentration of DMSO was used in the vehicle. For in vivo experiments, drugs were administered at 30mg/kg IP.
Gene Expression Profiling: RNA sequencing was performed on 100ng of hippocampal RNA obtained from mice after three days of daily, systemic injection of RGFP963, RGFP966, or vehicle. Results were validated with quantitative PCR (QPCR) using standard Taqman assays (Life Technologies).
©
2015 Macmillan Publishers Limited. All rights reserved.
Synaptogenesis Assay: Primary cortical neuron cultures were prepared from cortices dissected from P0 CD1 mice. Neurons were then seeded onto 96-well imaging plates (Corning) and were transfected with Synaptophysin-YFP plasmid constructs. ImageJ software (NIH) was used to quantify the relative number of fluorescent puncta obtained by imaging with the InCell 6000 at DIV10, relative to DIV8.
Electrophysiology: Whole-cell voltage clamp experiments were performed on pyramidal neurons from P0 hippocampal cultures. Drugs were added at DIV4 and recordings were made 18-24 hrs later. Miniature excitatory postsynaptic currents (mEPSCs) were isolated with 50µM picrotoxin, 1 µM tetrodotoxin and 50 µM DL-AP5 (Tocris), and recorded at -70 mV. Analysis of miniature events was performed using Clampfit 10.4 software (Molecular devices).
In Vivo Spine Imaging and Quantification: After 12 daily IP drug treatments, 11 week old Thy1GFP(m) mice were deeply anesthetized and perfused transcardially. Tissue was mounted onto microscope slides with an anti-fade reagent with DAPI (Prolong Gold, Life Technologies). For spine density and morphology, multiple branches emanating from 6-10 dendritic segments of eGFP+ CA1 oblique branches of pyramidal neurons that were ∼20–30 μm in length were imaged by Z-sectioning through the slice (from 25–75 μm from slice surface). Images were fast filtered with Image J. As segments were traced, each individual spine was marked. Only protuberances with a clear connection of the head of the spine to the dendritic shaft were counted as a spine. Pictures were visualized and elaborated with Neurolucida software (MicroBrightField).
Contextual Fear Conditioning: APP/PSI mice at least six months of age were given 12 daily IP injections of the appropriate compound or vehicle. At least 24 hrs after the final injection, mice
©
2015 Macmillan Publishers Limited. All rights reserved.
were placed into the training chamber and allowed to explore for 2.5 min, after which they received a single electric foot shock (2 s, 0.75mA). After the shock, the mice remained in the chamber for an additional 28 s before being returned to their home cage. To test for long-term memory (LTM), freezing was assessed 24 hrs later during a 5 min exposure to the context.
Statistical Analyses: One- or two-way univariate ANOVAs were applied to all data. Student’s ttests and post hoc tests were used when necessary. Significance was set at P200-fold more selective for HDAC3 than HDAC1 and HDAC2, with no activity detected at the other HDACs (Table S1) (Malvaez et al., 2013). We also investigated RGFP233, an inhibitor that targets HDACs 1, -2, -3 and -10, but with a ~75-fold greater selectivity for HDAC1 and HDAC2 (Table S1). Using this panel of HDAC-targeting compounds, we investigated how differing inhibition of HDAC isoforms impacted features of neuronal function, as well as memory in the context of a mouse model of AD.
©
2015 Macmillan Publishers Limited. All rights reserved.
To date, NaB represents the most selective HDACi (HDAC1, -2, -3, -8) known to be capable of driving synaptogenesis. Therefore, we first determined the impact of HDAC inhibition limited to HDAC1, -2 and -3 (RGFP963; RGFP968), HDAC1 and -2 (RGFP233) or HDAC3 alone (RGFP966) on synaptogenesis. Expression of YFP-tagged synaptophysin (SYN) in neurons reliably reports the presence of functional presynaptic terminals in cultured neurons (Harms et al., 2005). Therefore, we developed a live imaging synaptogenesis assay based on this reporter construct. We expressed the SYN-YFP fusion in young cortical neurons and then confirmed that the axonlocalized bright puncta colocalized with endogenous proteins expressed in synaptic vesicles (Fig. 2A). These data suggested that counting the number of SYN-YFP+ structures over time on a perneuron basis would provide a dynamic readout of synaptogenesis in developing cultured neurons. Based on this initial result, we designed an assay where neurons were imaged during a baseline session and then vehicle or the broad spectrum HDACi TSA was added to individual culture wells. Two days later, the same wells were re-imaged and the relative change in SYNYFP+ structures was calculated (Fig. 2B-C). There was an increase of ~15% in SYN-YFP+ puncta from DIV8 to DIV10 in the DMSO-treated wells. TSA-treated wells had a significantly greater increase over the same period (~50%; t(10)=-6.92, P
Neuropsychopharmacology www.neuropsychopharmacology.org
Methylphenidate modifies the motion of the circadian clock Lamotrigine in mood disorders and cocaine dependence Cortical glutamate in postpartum depression
Pharmacological Selectivity within Class I Histone Deacetylases Predicts Effects on Synaptic Function and Memory Rescue Gavin Rumbaugh, Stephanie E Sillivan, Emin D Ozkan, Camilo S Rojas, Christopher R Hubbs, Massimiliano Aceti, Mark Kilgore, Shashi Kudugunti, Sathyanarayanan V Puthanveettil, J David Sweatt, James Rusche, Courtney A Miller
Cite this article as: Gavin Rumbaugh, Stephanie E Sillivan, Emin D Ozkan, Camilo S Rojas, Christopher R Hubbs, Massimiliano Aceti, Mark Kilgore, Shashi Kudugunti, Sathyanarayanan V Puthanveettil, J David Sweatt, James Rusche, Courtney A Miller, Pharmacological Selectivity within Class I Histone Deacetylases Predicts Effects on Synaptic Function and Memory Rescue, Neuropsychopharmacology accepted article preview 3 April 2015; doi: 10.1038/npp.2015.93. This is a PDF file of an unedited peer-reviewed manuscript that has been accepted for publication. NPG are providing this early version of the manuscript as a service to our customers. The manuscript will undergo copyediting, typesetting and a proof review before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers apply.
Received 11 December 2014; revised 25 March 2015; accepted 31 March 2015; Accepted article preview online 3 April 2015
©
2015 Macmillan Publishers Limited. All rights reserved.
Pharmacological selectivity within Class I Histone Deacetylases predicts effects on synaptic function and memory rescue Gavin Rumbaugh1*#, Stephanie E. Sillivan1,2#, Emin D. Ozkan1, Camilo S. Rojas1, Christopher R. Hubbs1, Massimiliano Aceti1, Mark Kilgore3, Shashi Kudugunti4, Sathyanarayanan V. Puthanveettil1, J. David Sweatt3, James Rusche4, Courtney A. Miller1,2* 1
2
Department of Neuroscience and Metabolism & Aging, The Scripps Research Institute, Jupiter, FL USA 3 Department of Neurobiology, The Evelyn F. McKnight Brain Institute, The University of Alabama at Birmingham, Birmingham, AL USA 4 The Repligen Corporation, Waltham, MA USA
# Equal contribution *Correspondence: Gavin Rumbaugh Courtney A. Miller ([email protected]) ([email protected])
Running Title: Memory rescue by synaptogenic Class I HDAC inhibitors
©
2015 Macmillan Publishers Limited. All rights reserved.
ABSTRACT Histone deacetylases (HDACs) are promising therapeutic targets for neurological and psychiatric disorders that impact cognitive ability, but the relationship between various HDAC isoforms and cognitive improvement is poorly understood, particularly in mouse models of memory impairment. A goal shared by many is to develop HDAC inhibitors with increased isoform selectivity in order to reduce unwanted side effects, while retaining procognitive effects. However, studies addressing this tack at the molecular, cellular and behavioral level are limited. Therefore, we interrogated the biological effects of Class I HDAC inhibitors with varying selectivity and assessed a subset of these compounds for their ability to regulate transcriptional activity, synaptic function and memory. The HDAC 1, -2, -3 inhibitors, RGFP963 and RGFP968, were most effective at stimulating synaptogenesis, while the selective HDAC3 inhibitor, RGFP966, with known memory enhancing abilities, had minimal impact. Furthermore, RGFP963 increased hippocampal spine density, while HDAC3 inhibition was ineffective. Genome-wide gene expression analysis by RNA sequencing indicated that RGFP963 and RGFP966 induce largely distinct transcriptional profiles in the dorsal hippocampus of mature mice. The results of bioinformatic analyses were consistent with RGFP963 inducing a transcriptional program that enhances synaptic efficacy. Finally, RGFP963, but not RGFP966, rescued memory in a mouse model of Alzheimer’s Disease (AD). Together, these studies suggest that the specific memory promoting properties of Class I HDAC inhibitors may depend on isoform selectivity and that certain pathological brain states may be more receptive to HDAC inhibitors that improve network function by enhancing synapse efficacy.
©
2015 Macmillan Publishers Limited. All rights reserved.
INTRODUCTION Manipulation of gene transcription through epigenetic modifications to DNA and chromatin represents a promising and broad therapeutic avenue for the treatment of central nervous system disorders, including disorders of cognitive ability, such as Alzheimer’s Disease (AD). AD is marked by increasing dementia as neurodegeneration progresses, starting with the loss of synapses (Terry et al., 1991), which are fundamental components of the brain’s memory storage system. Epigenetics has received particular attention in recent years for its role in memory (Day and Sweatt, 2011) and hopes are particularly high for the potential of epigenetic modulation to drive cognitive enhancement and, perhaps, to rescue memory in AD (Abel and Zukin, 2008; Mikaelsson and Miller, 2011; Rudenko and Tsai, 2014).
Chromatin’s core histone proteins undergo posttranslational modification with memory, including acetylation, providing some molecular clues to how these enzymes regulate cognition. Transcriptionally permissive histone acetylation is regulated by histone acetyltransferases (HATs), which add acetyl moieties and histone deacetylases (HDACs), which remove them. There are five classes of transcriptionally repressive HDACs. Class I (HDAC1, 2, 3 and 8), Class IIA (HDAC 4, 5, 7 and 9), Class IIB (HDAC 6, 10), and Class IV (HDAC11) are zinc-dependent, whereas Class III sirtuins (SIRT1-7) are NAD-dependent and can also participate in non-histone protein acetylation. Numerous labs have now demonstrated that pharmacologic inhibition of HDACs, which leads to an accumulation of lysine acetylation and transcriptional activation, is capable of modulating memory (Graff and Tsai, 2013; Levenson et al., 2004). This has driven an excitement in the field for the potential of memory rescue in the face of disorders characterized by widespread synapse loss and memory impairments. However, the eleven non-sirtuin HDACs are involved in a myriad of processes throughout the body, leading many to propose that increasing HDACi isoform
©
2015 Macmillan Publishers Limited. All rights reserved.
selectivity may improve specificity for a given brain disorder and simultaneously decrease unwanted off-target effects (Graff and Tsai, 2013; Haggarty and Tsai, 2011; Mikaelsson and Miller, 2011). This naturally begs the question of which isoform(s) represent the ideal target for treating memory disorders.
Class I HDACs have received the most attention in relation to cognition. For instance, genetic, embryonic deletion of HDAC2, but not HDAC1, was found to enhance memory in wildtype mice and furthermore, viral-mediated knockdown of HDAC2 in the hippocampal CA1 region was sufficient to rescue memory in a mouse model of neurodegeneration (Graff et al., 2012; Guan et al., 2009). Similar memory enhancement was found following postnatal forebrain deletion of HDAC2 using a CAMKII-Cre driver (Morris et al., 2013). Likewise, focal deletion of HDAC3 accomplished through viral delivery of Cre into the hippocampus of healthy adult HDAC3 floxed mice enhanced memory (McQuown et al., 2011), though the role of this isoform has not been tested in mouse models of cognitive dysfunction. While broad genetic deletion of an HDAC may be accompanied by various developmental compensations and is therapeutically impractical, more temporally restricted manipulations of HDAC activity can be achieved through pharmacologic targeting with small molecules. These manipulations may also be more specific compared to genetic deletion because they target enzymatic activity, rather than deleting the protein. There are at least three classes of engineered HDACi’s: carboxylic acids (e.g. sodium butyrate [NaB], sodium phenylbutryate [SPB]), hydroxamic acids (e.g. trichostatin A [TSA], suberoylanilide hydroxamic acid [SAHA] and crebinostat), and ortho-aminoanilines or benzamides (e.g. RGFP136). However, a pharmacologic approach to HDAC inhibition has historically meant sacrificing isoform selectivity. For instance, TSA, which can enhance memory when delivered to a healthy brain (Blank et al., 2014; Fischer et al., 2007; Hawk et al., 2011;
©
2015 Macmillan Publishers Limited. All rights reserved.
Monsey et al., 2011), targets all 11 HDACs. SPB and valproic acid, HDACi’s with similarly broad activity, rescue memory in mouse models of AD (Kilgore et al., 2010; Ricobaraza et al., 2009). Additional insight into the role of individual HDACs in cognition can be gleaned from HDACi’s with greater isoform specificity. For example, the most commonly used HDACi for cognitive studies, NaB, was once thought to be a broad spectrum inhibitor, but has since been determined to have a surprising degree of selectivity for Class I HDACs (Kilgore et al., 2010). Further, SAHA and crebinostat’s activity are largely limited to Class I HDACs (1, 2, 3 and 8) and HDAC6. Acute administration of NaB, SAHA and crebinostat have all been shown to enhance memory in wildtype mice and to drive synaptogenesis (Blank et al., 2014; Fass et al., 2013; Fischer et al., 2007; Guan et al., 2009; Intlekofer et al., 2013; Levenson et al., 2004; Mahan et al., 2012). Further, NaB and SAHA rescue memory in mice with reduced cognitive ability, including AD models (Fischer et al., 2007; Kilgore et al., 2010). Two of the most selective HDACi’s used to date, RGFP136 and RGFP966, bear a high degree of selectivity for HDAC3 and enhance memory in wildtype mice (Malvaez et al., 2013; McQuown et al., 2011). When the results of these studies are taken together, it can readily be concluded that manipulating members of Class I HDACs (1, 2, 3 and 8) can improve memory, with genetic manipulations further pointing to the potential of HDAC2 and HDAC3 as targets for improving cognition in a “diseased” state. However, selective inhibitors of HDAC2 or HDAC3 have not been tested in mouse models of reduced cognitive function. Furthermore, the impact of selective inhibitors on synaptic function and spinogenesis, which are the neurobiological substrates believed to drive improved cognition in AD models (Knobloch and Mansuy, 2008; Nistico et al., 2012; Pozueta et al., 2013), remains unknown. Therefore, we assessed the impact of several new HDACi’s with differing Class I selectivity profiles at various neurobiological levels to better understand the relationship between HDACi isoform selectivity and regulation of cognitive function.
©
2015 Macmillan Publishers Limited. All rights reserved.
MATERIALS AND METHODS For additional details, please see Supplementary Materials and Methods.
Animals: 8-week old C57BL/6 mice were used for gene expression analyses (The Jackson Laboratory, Bar Harbor, ME). Thy1-GFP(m) (B6.Cg-Tg(Thy1-EGFP)MJrs/J; stock number 007788) and APPswe/PS1dE9 double transgenic mice (APP/PS1; B6C3-Tg (APPswe,PSEN1dE9)85Dbo/J; stock number 004462) (Jankowsky et al., 2003) were also obtained from Jackson Laboratory. P0 CD1 mice were used for electrophysiological recordings and synaptogenesis assays (CD1(ICR) (Strain Code 022), Charles River Laboratory, Hollister, CA). All procedures were performed in accordance with the Scripps Florida Research Institute at Jupiter, Florida Institutional Animal Care and Use Committee and with national regulations and policies.
Drugs: Trichostatin A (TSA; Tocris) was dissolved in DMSO at a concentration of 300mM. RGFP966, RGFP963, and RGFP233 were provided by Repligen Corporation. The drugs were dissolved in DMSO and diluted in 30% HPBCD, 0.1M acetate, pH 5.5. The final DMSO concentration was no greater than 5% and the same concentration of DMSO was used in the vehicle. For in vivo experiments, drugs were administered at 30mg/kg IP.
Gene Expression Profiling: RNA sequencing was performed on 100ng of hippocampal RNA obtained from mice after three days of daily, systemic injection of RGFP963, RGFP966, or vehicle. Results were validated with quantitative PCR (QPCR) using standard Taqman assays (Life Technologies).
©
2015 Macmillan Publishers Limited. All rights reserved.
Synaptogenesis Assay: Primary cortical neuron cultures were prepared from cortices dissected from P0 CD1 mice. Neurons were then seeded onto 96-well imaging plates (Corning) and were transfected with Synaptophysin-YFP plasmid constructs. ImageJ software (NIH) was used to quantify the relative number of fluorescent puncta obtained by imaging with the InCell 6000 at DIV10, relative to DIV8.
Electrophysiology: Whole-cell voltage clamp experiments were performed on pyramidal neurons from P0 hippocampal cultures. Drugs were added at DIV4 and recordings were made 18-24 hrs later. Miniature excitatory postsynaptic currents (mEPSCs) were isolated with 50µM picrotoxin, 1 µM tetrodotoxin and 50 µM DL-AP5 (Tocris), and recorded at -70 mV. Analysis of miniature events was performed using Clampfit 10.4 software (Molecular devices).
In Vivo Spine Imaging and Quantification: After 12 daily IP drug treatments, 11 week old Thy1GFP(m) mice were deeply anesthetized and perfused transcardially. Tissue was mounted onto microscope slides with an anti-fade reagent with DAPI (Prolong Gold, Life Technologies). For spine density and morphology, multiple branches emanating from 6-10 dendritic segments of eGFP+ CA1 oblique branches of pyramidal neurons that were ∼20–30 μm in length were imaged by Z-sectioning through the slice (from 25–75 μm from slice surface). Images were fast filtered with Image J. As segments were traced, each individual spine was marked. Only protuberances with a clear connection of the head of the spine to the dendritic shaft were counted as a spine. Pictures were visualized and elaborated with Neurolucida software (MicroBrightField).
Contextual Fear Conditioning: APP/PSI mice at least six months of age were given 12 daily IP injections of the appropriate compound or vehicle. At least 24 hrs after the final injection, mice
©
2015 Macmillan Publishers Limited. All rights reserved.
were placed into the training chamber and allowed to explore for 2.5 min, after which they received a single electric foot shock (2 s, 0.75mA). After the shock, the mice remained in the chamber for an additional 28 s before being returned to their home cage. To test for long-term memory (LTM), freezing was assessed 24 hrs later during a 5 min exposure to the context.
Statistical Analyses: One- or two-way univariate ANOVAs were applied to all data. Student’s ttests and post hoc tests were used when necessary. Significance was set at P200-fold more selective for HDAC3 than HDAC1 and HDAC2, with no activity detected at the other HDACs (Table S1) (Malvaez et al., 2013). We also investigated RGFP233, an inhibitor that targets HDACs 1, -2, -3 and -10, but with a ~75-fold greater selectivity for HDAC1 and HDAC2 (Table S1). Using this panel of HDAC-targeting compounds, we investigated how differing inhibition of HDAC isoforms impacted features of neuronal function, as well as memory in the context of a mouse model of AD.
©
2015 Macmillan Publishers Limited. All rights reserved.
To date, NaB represents the most selective HDACi (HDAC1, -2, -3, -8) known to be capable of driving synaptogenesis. Therefore, we first determined the impact of HDAC inhibition limited to HDAC1, -2 and -3 (RGFP963; RGFP968), HDAC1 and -2 (RGFP233) or HDAC3 alone (RGFP966) on synaptogenesis. Expression of YFP-tagged synaptophysin (SYN) in neurons reliably reports the presence of functional presynaptic terminals in cultured neurons (Harms et al., 2005). Therefore, we developed a live imaging synaptogenesis assay based on this reporter construct. We expressed the SYN-YFP fusion in young cortical neurons and then confirmed that the axonlocalized bright puncta colocalized with endogenous proteins expressed in synaptic vesicles (Fig. 2A). These data suggested that counting the number of SYN-YFP+ structures over time on a perneuron basis would provide a dynamic readout of synaptogenesis in developing cultured neurons. Based on this initial result, we designed an assay where neurons were imaged during a baseline session and then vehicle or the broad spectrum HDACi TSA was added to individual culture wells. Two days later, the same wells were re-imaged and the relative change in SYNYFP+ structures was calculated (Fig. 2B-C). There was an increase of ~15% in SYN-YFP+ puncta from DIV8 to DIV10 in the DMSO-treated wells. TSA-treated wells had a significantly greater increase over the same period (~50%; t(10)=-6.92, P
